Expression of TRP channels and ANOs in normal human epidermal keratinocytes
The levels of endogenous gene expression of TRP channels and ANOs in NHEKs are unclear. To clarify the expression patterns, we performed RT-PCR analysis of cultured NHEKs (Fig. 1A). Previous reports suggested that TRPV1, TRPV3, TRPV4, TRPV6 proteins were expressed in keratinocytes.17 Here, we observed mRNAs of TRPV1, TRPV2, TRPV3, TRPV4, TRPV6, ANO1, ANO4, ANO9 and ANO10 (Fig. 1A). Although ANO2 also functions as a CaCC, a discrete band with the predicted molecular weight was not detected in NHEKs. In contrast, ANO1 protein expression was observed by Western blotting (Fig. 1B). These results suggested significant expression of ANO1 in NHKEs. Additionally, we performed calcium-imaging experiments using Fura-2 to investigate functional expression of TRP channels. Whereas camphor (10 mM, a TRPV3 agonist) and GSK1016790A (300 nM, a TRPV4 agonist) obviously induced intracellular calcium increases in all cells, capsaicin (300 nM to 3 µM, a TRPV1 agonist), menthol (100 µM, a TRPM8 agonist), allyl isothiocyanate (AITC, 100 µM or 1 mM, a TRPA1 agonist), probenecid (100 µM, a TRPV2 agonist) or 1-oleoyl-acetyl-sn-glycerol (OAG, 90 µM, a TRPC6 agonist) did not produce clear responses (Fig. 1C).
We also performed calcium-imaging experiments using calcium-free extracellular medium because intracellular calcium concentrations are supposed to be reduced upon removal of extracellular calcium in cells expressing TRPV6, which can be constitutively active. However, the intracellular calcium concentrations were not different in the presence or absence of extracellular calcium (Fig. 1D). In addition, a typical TRPV6-mediated current with inward rectification was not observed in NHEKs (Fig.1E). These results indicated that in NHEKs, the most active TRP channels were TRPV3 and TRPV4. However, expression of other TRP channels was suggested in RT-PCR experiments. Thus, ANO1 could be activated by calcium influx through TRPV3 or TRPV4 in cells co-expressing these ion channels. Therefore, we decided to focus on the interaction between TRPV3 and ANO1 in this study because their interaction had not received attention in the literature.
TRPV3-ANO1 interaction in HEK293T cells
We performed whole cell patch-clamp experiments using HEK293T cells heterologously expressing TRPV3 and ANO1 to investigate their functional interaction. NMDG-Cl bath and pipette solutions were used to identify chloride currents through ANO1 because NMDG is known not to permeate pores of cation channels. We used camphor as a TRPV3 agonist since a previous report showed other TRPV3 agonists, 2-APB and carvacrol, inhibited ANO1 currents18. Under these conditions, chloride currents were clearly observed in cells expressing both human TRPV3 (hTRPV3) and human ANO1 (hANO1), but not in cells expressing hTRPV3 or hANO1 alone (Fig. 2A, B). The currents were interpreted to be chloride currents passing through hANO1 that had been activated by calcium entering cells through hTRPV3. The currents were observed even with intracellular 1, 2-bis (o-aminophenoxy) ethane-N,N,N’,N’-tetraacetic acid (BAPTA) (5 mM), which is a relatively strong calcium chelator. This result suggested that TRPV3-ANO1 interaction could occur in a local calcium nanodomain in which BAPTA-mediated calcium chelation did not function. Since previous studies suggested that both TRPV1 and TRPV4 physically interacted with ANO12,3,19, we performed immunoprecipitation and Western blotting experiments using anti-ANO1 and anti-TRPV3 antibodies with extracts from HEK293T cells (Fig. 2C). TRPV3 and ANO1 proteins were co-immunoprecipitated in cells expressing both proteins while there were no TRPV3 bands in the extracts from cells transfected with hANO1 cDNA, hTRPV3 cDNA or pcDNA3.1 plasmid alone, indicating the physical interaction of hTRPV3 with hANO1. These results suggested functional and physical interaction between hTRPV3 and hANO1 in the heterologous expression system.
TRPV3-ANO1 interaction in NHEKs
Intracellular calcium increases were observed in all NHEKs upon camphor application (Fig. 1C). Therefore, we performed whole-cell patch-clamp experiments in NHEKs. Camphor-induced chloride currents were observed in 148 mM chloride-containing bath solution (Fig. 3A). The reversal potential of the chloride currents was shifted to a positive direction when the extracellular chloride concentration was changed to 4 mM (Fig.3B, C). That result indicated that chloride was a major ion carrier of the camphor-induced currents. The release of calcium from the endoplasmic reticulum might not be a major contributor to increases in intracellular calcium concentrations in NHEKs because the increases in intracellular calcium concentrations were small in the calcium-free bath solution (Fig.3D). This interpretation was confirmed in the patch-clamp experiments in which the camphor-induced currents were very small in the extracellular calcium-free solution (Fig. 3E). Therefore, TRPV3 agonist-induced chloride currents through ANO1 in NHEKs mainly depend on the calcium influx through TRPV3 from extracellular regions. The camphor-induced chloride currents were inhibited by Ani9, a strong ANO1 inhibitor (Fig. 3F), further supporting the TRPV3-ANO1 interaction in NHEKs.
Effects of an ANO1 inhibitor or low chloride medium on NHEK cell migration/proliferation
Previous studies showed that TRPV3 contributes to itch and warmth sensations and wound healing by keratinocytes5-8. It has been strongly suggested that TRPV3 activation accelerates wound healing in the oral cavity5. Moreover, the basic histological properties of the oral cavity are similar to those of skin compared to other mucosa in the body20. Furthermore, ANO1 could be involved in tissue development after birth21, and it is well-known to be a positive regulator of migration and proliferation in cancer cells10,11,14,15. Therefore, we hypothesized that TRPV3-ANO1 interaction might affect the migration and/or proliferation of NHEKs and the process of wound healing.
To investigate the involvement of ANO1 in wound healing, we analyzed the effects of another ANO1 blocker, T16Ainh-A01 (T16A). The assessment incorporated a culture insert to quantitate cell activity (Fig. 4). In these experiments, NHEKs were cultivated within a culture insert to almost 100% confluency in which cells migrated to spaces between cell clusters22 (Fig. 4A). NHEKs usually migrated to the open spaces, an area separated by the insert, for approximately 12 h after the insert was removed. In this way, migration and proliferation filled the area by about 80% within 24 h. However, cell migration and/or proliferation in T16A (5 µM)-containing medium was obviously inhibited without affecting ANO1 protein levels (Suppl. Fig. 1). Importantly, the inhibition was lost after the washout of T16A (Fig. 4B, C). Those observations suggested that the T16A effect was not due to cell-death or irreversible cell damage. In addition, we analyzed cell migration velocity using time-lapse imaging with a confocal microscope and cell proliferation using an MTT assay (Fig. 4D-F). Migration velocity was reduced after T16A application, and the reduction lasted throughout the inhibition of ANO1 (Fig. 4D). Moreover, the migration velocity recovered to the initial level after washout of T16A (Fig. 4D and E). Cell proliferation was also reduced by T16A application (Fig. 4F). These results suggested the importance of chloride ions for cell migration and proliferation. Therefore, we performed an assay with a culture insert in low chloride medium (Fig. 5). Intracellular chloride concentrations should be reduced upon depletion of extracellular chlorides23. After removal of the culture insert, the filled areas were drastically reduced in the low chloride-containing medium, an effect that was lost after the change back to the control medium (Fig. 5). These results indicated that chloride flux through ANO1 plays critical roles in cell migration and/or proliferation.
Direction of chloride movement through chloride channels in NHEKs
Although the previous results suggested the importance of chloride ions for cell migration and/or proliferation, the actual roles of chloride ions in keratinocytes are largely unknown. To address this question, we attempted to determine the direction of chloride movement. Chloride permeation through chloride channels depends on intracellular chloride concentrations and membrane potentials. Although chloride channel function could affect intracellular chloride concentrations, they should be maintained by the function of several chloride transporters24. Therefore, we examined the expression patterns of chloride transporters, including Na-K-Cl cotransporters (NKCCs) and K-Cl cotransporters (KCCs) using RT-PCR. mRNA expression of the genes coding for NKCC1, KCC1, KCC2, KCC3 and KCC4 was suggested (Fig. 6A). KCC2 is a neuron-specific KCC, and intracellular chloride concentrations are kept at a low level through chloride efflux by KCC2 in cells in the central nervous system, and opening of the chloride-permeable channels causes chloride influx. Therefore, we performed a chloride-imaging experiment using a chloride indicator, MQAE25-27. The calculated intracellular chloride concentrations of NHEKs were relatively low (6.8 ± 1.3 mM) (Fig. 6B,C), which is consistent with KCC2 expression at least at the mRNA level in NHEKs. The calculated equilibrium potential for chloride ions (-75.7 mV) suggests that chloride influx occurred through ANO1 in NHEKs at the reported resting membrane potentials of skin keratinocytes (-24 to -40 mV) 28-30.
An ANO1 inhibitor induces MAP kinase phosphorylation
Previous studies suggested that low intracellular chloride concentrations induce the phosphorylation of mitogen-activated protein kinase (MAPK), although its precise mechanisms are not well known. MAPK cascades are involved in the life and death of many cells31,32 (Fig. 7A). For instance, extracellular signal-related kinase (ERK), which is phosphorylated by MAPK kinase (MKK)1/2, is involved in cell proliferation and differentiation. On the other hand, p38 and c-Jun N-terminal kinase (JNK), which are phosphorylated by MKK3/4/6 and MKK4/7, respectively, induce cell cycle arrest and apoptosis. Hence, we investigated MAPK phosphorylation using a Western blot method (Fig. 7B, C). An ANO1 inhibitor, T16A, increased phosphorylation of p38, but not that of ERK or JNK. These results suggested that ANO1 is involved in cell cycle arrest and/or apoptosis. However, ANO1 inhibition did not induce cell death in the culture insert assay based upon the fact that cell appearance visualized with a calcein-AM staining was not affected by ANO1 inhibition (Fig. 4). In addition, there were no effects of T16A treatment on the expression of differentiation-related genes, including KRT1, IVL and TGM1, in both differentiated and undifferentiated conditions (Suppl. Fig. 2). This result is consistent with the lack of effects of the T16A treatment on ERK phosphorylation (Fig. 7B), which is known to be related to differentiation (Fig. 7A). Therefore, we decided to focus on cell cycle arrest.
An ANO1 inhibitor induced cell cycle arrest during the culture insert assay
Because the MAPK analyses suggested cell cycle arrest by ANO1 inhibition, we performed a cell cycle assay by using a redox dye (Fig. 8). Redox conditions are closely related to the cell cycle33. For instance, intracellular redox conditions in cells in the G0/G1 phases are relatively reductive, whereas redox conditions are gradually shifted to more oxidative ones upon progression to G2/M phases. In this assay system, cells in G0/G1 phase, S phase and G2/M phases can be visualized as yellow-green, green and dark blue, respectively (Fig. 8A). To clarify the color variation, each cell was visualized as a red color depending on signal levels (Fig. 8B). T16A treatment increased cell populations in G0/G1 phases and reduced cell populations in S phase during the culture insert assay (Fig. 8B, C). This result indicated that cell cycle progression from G0/G1 to S phase was suppressed by ANO1 inhibition.